Abstract [en]

Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography.

Morin Zetterberg, Malin

Abstract [en]

Polyethylene glycol-stabilized lipodisks have emerged as a novel type of lipid-based nanoparticles with high potential as both drug carriers and biomimetic membranes. In this thesis we assess both of these applications, and show how the properties of the lipodisks can be further developed and optimized.

Initially, we show that the antimicrobial peptides melittin, alamethicin and magainin 2, in spite of their very different physico-chemical properties and suggested modes of action on membranes, all have high affinity to lipodisks. Using melittin as a model peptide, we confirm a maintained antimicrobial effect of disk-formulated peptides. We also show that melittin dissociates slowly from the disks, resulting in extended drug release and prolonged antibacterial effect. Additionally, we present evidence that the peptide is protected against enzymatic degradation when formulated in the disks.

Further, we develop a stable HPLC-MS system with immobilized lipodisks as model membranes. The stability of the system is confirmed by drug partitioning analysis using 15 different drug compounds. We also show how the lipodisk column can be supplemented with cyclooxygenase by in situ incorporation of the protein in the lipodisks. The specific binding of the protein to the disks is confirmed using QCM-D.

Finally, by changing the polymer length and applying a new preparation protocol, we have optimized the lipodisks for use as drug carriers and biomimetic membranes. Previous lipodisk studies have been conducted on systems containing PEG-lipids with polymer molecular weights of 2000 or 5000 Da. Also, conventional protocols for the preparation of lipodisks typically require a PEG-lipid concentration of 15 mol% or more. Here we show that stable lipodisks can also be produced using PEG-lipids with a 1000 Da molecular weight polymer and that the use of shorter PEG-lipids dramatically improve the amount of lipodisks that can be immobilized on silica surfaces. Moreover, through the development of a method in which lipid mixtures are sonicated at low temperatures, we produce lipodisks containing as little as 2 mol% PEG-lipid. We present data verifying that these disks are superior to disks with higher PEG-lipid content in terms of their ability to incorporate externally added PEG-lipids functionalized with targeting agents.